Replicative senescence, the finite replicative limit of human diploid fibroblasts
Biological aging, an inevitable process common to multicellular organisms, involves a progressive physiological decline and associated pathologic degeneration of tissues and organs. The fundamental basis of aging remains enigmatic (Goldstein, 1992; Dice, 1993). The discovery that human diploid fibroblasts (HDF) have a finite proliferative lifespan opened the way to explore aging at the cellular level (Hayflick, 1965). The major feature of senescent HDF is their inability to synthesize DNA due to G1 arrest and failure to traverse the G1/S boundary (Goldstein, 1990; Cristofalo and Pignolo, 1993). A further hallmark of senescence is the dominant effect of the senescent nucleus on DNA synthesis in the young nucleus, as demonstrated in experiments involving somatic cell fusions between young and old cells. Initiation of DNA synthesis in the young HDF nucleus was extinguished, but ongoing DNA synthesis was not (Norwood et al., 1974; Stein and Yanishevsky, 1981). Moreover, this effect is abrogated by blockers of RNA and protein synthesis indicating that inhibition of DNA synthesis depends upon one or more proteins and perhaps on a direct inhibitory action of RNA(s) (Norwood et al., 1990).
Human diploid fibroblast cells (HDF) cultured in vitro provide an excellent model system for the study of biologic aging (Hayflick 1965; Goldstein 1990). These cells possess a limited replicative lifespan ("senescence in vitro"), that can be measured as the MPD.sub.max, the maximum number of Mean Population Doublings accruing until phaseout. However, the great majority of senescent cells remain viable and capable of carrying out all metabolic and macromolecular functions except semiconservative DNA synthesis.
In several large series of HDF cultures, the MPD.sub.max is inversely proportional to the age of the donor (reviewed in Goldstein 1989). Moreover, HDF from subjects with Werner syndrome (WS, see below for discussion) display a sharply curtailed growth capacity compared to age-matched controls (Thweatt, et al. 1993). Thus, physiologic rather than chronologic age determines the MPD.sub.max, and HDF clearly count cell divisions, rather than calendar or metabolic time, to a critical limit (Goldstein 1990; Goldstein 1989). That the replicative lifespan of cultured fibroblasts from a diversity of animal species is directly proportional to the maximum life expectancy of these species (ranging from two years to 150 years) indicates the presence of powerful genetic determinants of cellular senescence (Goldstein 1990; Goldstein 1992). Taken together, the data suggest a critical connection between senescence of HDF in vitro and biologic aging in vivo.
Dominance of the senescent phenotype in HDF
Cell fusion experiments have guided the search for root causes of HDF senescence. In repeated attempts at forming proliferating cell hybrids, young HDF (yHDF) failed to rescue senescent HDF (SHDF) after cell fusion, but permanent lines were able to do so (Goldstein, 1971). In short-term cell hybrids containing a senescent and a young nucleus within a single cytoplasm, i.e. heterocaryons, initiation of DNA synthesis in the yHDF nucleus was extinguished (Goldstein, 1971) but ongoing DNA synthesis was not (Yanishevsky, et al. 1980; reviewed in Norwood, et al. 1990). Brief post-fusion treatment of such heterocaryons with blockers of RNA and protein synthesis abrogated the inhibition (Norwood, et al. 1990).
Taken together, these data indicate that senescence is a dominant trait mediated by proteins or perhaps RNAs. In strong support of this concept, Lumpkin, Smith and coworkers microinjected polyA.sup.+ RNA from sHDF into yHDF and were able to inhibit DNA synthesis (Lumpkin, et al. 1986).
Relationship between HDF senescence and negative growth regulation
The primary mechanism by which senescent cells irreversibly lose the ability for transit through the G1/S checkpoint of the cell cycle, which differentiates them from growth arrested (quiescent) cells, is yet to be discovered. It is obvious that quiescence (arrested) and senescence share many proteins in common whose activity lead to the inhibition of DNA synthesis. Recently discovered proteins controlling cell cycle progression belong to this category. Their function is to inhibit activity of cyclin dependent kinase-cyclin (CDK-cyclin) complexes. These proteins are termed CDK inhibitory proteins (CKIs) and appear to be responsible for braking the cell cycle. Some of these proteins are activated in response to extracellular signals, while others appear to function intrinsically during the cell cycle (reviewed in Hunter 1993 and Peters, et al. 1994).
The p21 protein was initially identified by functional cloning of a gene sequence (SDI1) coding for an inhibitor of DNA synthesis and is overexpressed in sHDF at a level approximately 10-20 times the level seen in yHDF (Noda, et al. 1994). The identical protein was discovered virtually simultaneously by three other laboratories investigating systems unrelated to senescence, p21 and CIPI were isolated by their ability to bind and inhibit Cdk2-cyclin A and Cdk2-cyclin E complexes activities (Xiong, et al. 1993; Harper 1993), and WAF1 was induced by p53 protein in response to DNA damage, leading to transient cell cycle arrest by inhibiting CDKs (El-Deiry, et al. 1993; Dulic, et al. 1994).
Another negative regulator of cell cycle transit named p16, identified by its association with Cdk4 in the yeast two-hybrid protein interaction system, appears to specifically inhibit Cdk4-cyclin D kinase activity in vitro (Serrano, et al. 1993). A major target of this kinase seems to be the retinoblastoma product (Rb), which must be phosphorylated for proper progression through GI phase. Available data support the proposal that p16 prevents phosphorylation of Rb (Serrano, et al. 1993). Closely related studies, primarily by Stein and co-workers, have analyzed the role of Rb in HDF senescence. Following serum stimulation Rb remains underphosphorylated in sHDF, in contrast, phosphorylated Rb is abundant following serum stimulation of quiescent (arrested) yHDF (Stein, et al. 1990). Moreover, underphosphorylated Rb in sHDF is associated with the failure to express Cdc2, cyclin A and cyclin B (Stein, et al. 1991; Richter, et al. 1991), the inability to phosphorylate the Cdk2-cyclin E complex (despite its elevated protein level), and the attenuation of Cdk2-cyclin D1 and Cdc2-cyclin A complexes activities (Dulic, et al. 1993). The intrinsic cell cycle machinery is controlled by external signals such as growth factors and antimitogens which allows for coordination of cell division with environmental and developmental stimuli. TGF-.beta. which can exhibit antimitogenic activity (Moses, et al. 1990) is known to play a role in expression of certain mRNAs and proteins like fibronectin, .alpha.(I)collagen, thrombospondin and SPARC/osteonectin (Penttinen, et al. 1988; Reed, et al. 1994), which are overexpressed in sHDF and WS HDF (Murano, et al. 1991), and also has been associated with the inhibition of the Cdk2-cyclin E complex kinase activity (Koff, et al. 1993). The protein responsible for this inhibition, p27, recently has been identified as associated with the Cdk2-cyclin E complex in cells arrested by TGF-.beta. (Polyak, et al. 1994; Polyak, et al. 1994; Toyoshima, et al. 1994). p27 also appears to be involved in cell cycle arrest imposed by contact inhibition (Polyak, et al. 1994).
Transcription factors and their role in senescence
Senescing cells undergo changes which suggest altered transcriptional regulation of gene expression. Because transcription factors are attractive candidates which may ultimately specify the senescent phenotype, many studies have been performed to describe the expression and activity of known transcription factors in senescent cells. These studies revealed that E2F transcription factor which is a positive regulator of several late G1 phase genes required for G1/S transition, is underexpressed in senescent cells and its activity is negatively regulated by the unphosphorylated form of Rb (Dimri, et al. 1994; Nevins 1992; Flemington, et al. 1993). Moreover in sHDF genes coding for transcription factors involved in the immediate early response to growth factors such as c-fos, Id-1h and Id-2h, appear to be irreversibly repressed (Dimri, et al. 1994; Seshadri, T. et al. 1990; Riabowol, et al. 1992; DeTata, et al. 1993; Hara, et al. 1994) or their binding activity is changed (Dimri, et al. 1994). However, there is a paucity of information about transcription factors as positive regulators of genes involved in inhibition of DNA synthesis and cell proliferation. Indeed a transcription factor specific for or overexpressed in senescent cells, has yet to be identified.
LIM proteins--a new family of transcription regulators
An important new family of proteins, the LIM protein family, has recently been described with roles in developmental and cell growth regulation. The LIM protein family, named for three of the originally identified protein members, lin-11 (Freyd, et al. 1990), isl-1 (Karlsson, et al. 1990), and mec-3 (way, et al. 1988), is defined by the presence of one to three repeats of a 52-residue segment containing two adjacent zinc binding domains separated by a two-residue linker (CX.sub.2 CX.sub.17 HX.sub.2 C)--X.sub.2 --(CX.sub.2 CX.sub.17 CX.sub.2 C/H/D). Although the LIM domain consists of two "zinc finger" domains, a controversy still remains about its DNA binding activity (Sanchez-Garcia, et al. 1994). Several studies indicate that it serves rather as a protein binding interface (Schmeichel, et al. 1994).
The LIM family consists of a variety of proteins with diverse functions and subcellular distributions; it includes transcription factors, protooncogene products and components of adhesion plaques. Based on the protein structure one can categorize the LIM family into three different groups. First, proteins containing a DNA binding homeodomain and a transcription activation domain adjacent to the LIM domains. This subfamily includes transcription factors involved in cell fate determination and differentiation as lin-11, isl-1 and mec-3. The second group, named "LIM-only" proteins, consists of several members that do not contain any additional known functional domains except LIM domains. LIM-only proteins appear to be involved in the regulation of gene activity even if they do not bind to DNA themselves. This group includes among others the protooncogene rhombotin-1, focal adhesion protein zyxin, cysteine-rich intestinal protein CRIP (Sanchez-Garcia, et al. 1994) and three newly discovered proteins with roles in the control of cell proliferation. MLP--muscle LIM protein plays a role in muscle differentiation by driving undifferentiated cells out of the cell cycle, a crucial step for initiation of the differentiation process (Arber, et al.). The protein ril was isolated from a revertant of ras-transformed cells and seems to be involved in the maintenance of normal cell growth (Kiess, et al. 1995). This gene is expressed in a variety of normal differentiated cells but is down-regulated in ras-transformed cells suggesting its function as a negative growth regulator. Another member of the LIM-only group, hic-5 protein was originally isolated from a mouse osteoblastic cell line whose growth was inhibited by TGF-.beta.1 (Shibanuma, et al. 1994). Hic-5 expression is also repressed in ras-transformed fibroblasts as well as in several cell lines established from human tumors. On the other hand the level of its transcript accumulates during senescence in vitro and its overexpression driven by the cytomegalovirus promoter suggests that hic-5 has a cytostatic effect on cell growth (Shibanuma, et al. 1994).
Third, a recently described group of proteins which in addition to LIM domains also contain a protein kinase activity, is represented by two members: Kiz-1, with a role in cell proliferation and neuron differentiation (Bernard, et al. 1994), and LIMK specific for lung tissue (Ohaski, et al.). The specific function for both proteins is not yet known, but there is evidence for their nuclear localization.
Differential gene expression during cellular senescence
Werner syndrome (WS) provides an excellent model for the study of aging because it is a genetically-determined syndrome with features of premature aging (Thweatt, et al. 1993; Goldstein 1978; Salk 1982). The multifaceted pathology that occurs sporadically during aging of normal persons appears almost universally in WS subjects, which becoming manifest earlier and with greater severity. Without exception, HDF derived from WS subjects display a curtailed replicative lifespan and also yield a dominant inhibition of DNA synthesis in hybrid cell fusions with normal yHDF (Salk 1982; Tanaka, et al. 1980). The in vitro observations lead to the prediction that the genes responsible for inhibition of DNA synthesis should be overexpressed in WS cells (Murano, et al. 1991; Goldstein, et al. 1989).